Abstract

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Interaction between the N- and C-terminal domains is essential for the solubility of heterologous SUFU. (a) Interactions between residues in the five-helix bundle formed between the two domains. (b) Size-exclusion chromatography profiles; the open and filled arrows indicate peaks corresponding to aggregated protein eluted with the void volume and soluble monomeric protein, respectively.

The SUFU IDR has distinct structural properties. (a) Thermal stability assays of MBP-SUFU constructs performed in triplicate, either alone (blue) or with GLI1p (red) or GLI1p-SH (green). All constructs bind to GLI1p; however, MBP-SUFU-FL has different physical properties upon initial GLI1p binding, as shown by the marked increase in fluorescence. (b) Microscale thermophoresis experiments with FAM-GLI1p and titrated MBP-SUFU constructs, showing an average of three separate experiments. All proteins have similar affinity, but the thermophoretic properties of FAM-GLI1p are modified differently between the MBP-SUFU-FL construct and the MBP-SUFU-Δ and MBP-SUFU-SH constructs, reflecting a difference in shape.

Crystal structure of MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A in complex with GLI3p. (a) Superposition of the N-terminal domains of apo (yellow with grey linker) and peptide-bound (beige, N-­terminal domain; green, C-terminal domain; purple linker) structures shows a 58° rotation of the C-terminal domain via a flexible linker. (b) The position of GLI3p in the MBP-216H_220H_-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A–peptide co-crystal. An averaged kick OMIT map contoured at 3.0σ shows well defined density for GLI3p lying between the β-­sheets of the SUFU N-terminal domain (beige) and C-terminal domain (green).

MBP-SUFU and GLI peptides interact in the same way in solution as in crystals. (a) HDX analysis of MBP-SUFU-FL with GLI3p (blue). Areas that are more protected from exchange in the presence of the peptide are highlighted in pink. (b) Comparison of SAXS envelopes for MBP-SUFU-Δ in the absence (left) and presence (right) of GLI1p. Crystal structures of apo MBP-SUFU-Δ and of MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A co-crystallized with GLI3p superpose well on apo and holo SAXS envelopes, respectively.